This application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 60/480,536 filed Jun. 20, 2003, which is hereby incorporated by reference in its entirety.
1. Field of the Invention
This invention was made with government support under grant number 99-CRHF-0-6055 awarded by the United States Department of Agriculture/CREES. The government has certain rights in the invention.
The present invention relates generally to the fields of microbiology and enzymology. More particularly, it concerns methods and compositions involving endopeptidase enzymes from bacteria that cleave peptides, particularly bitter peptides and peptides involved in gluten inflammation. In some embodiments, methods and compositions concern PepO2 and/or PepO3, and their use in reducing bitterness in foods, such as cheese, or treating or preventing celiac sprue.
2. Description of Related Art
Lactobacillus helveticus belongs to a group of organisms known as lactic acid bacteria (LAB), which are defined by the production of lactic acid as a major product of carbohydrate fermentation. Lb. helveticus has multiple amino acid (AA) auxotrophies and thus is dependent on transport of AA and/or transport and hydrolysis of exogenous peptides to satisfy these nutritional requirements. In AA defined media, Lb. helveticus CNRZ32 can grow without Ala, Asn, Cys, Gln, Gly, and Ser when they are absent individually (Christensen, 2000). The fermentation of Bos taurus milk is a common system to study the proteolytic system and physiology of Lb. helveticus, providing a relatively consistent environment and well characterized set of proteins as a starting point, as well as having adaptive significance for dairy related LAB. Since all of the identified peptidases of L. helveticus are believed to be intracellular, the acquisition of AA is also likely to be dependent on the activity of at least one extracellular proteinase capable of hydrolyzing caseins into transportable peptides (Kunji et al., 1996). Therefore, obtaining AA through the hydrolysis of caseins (the preferentially hydrolyzed milk proteins) requires a complex proteolytic system comprised of proteinase(s), endopeptidase(s), aminopeptidase(s), tripeptidase(s), dipeptidase(s), and peptide transport systems (Christensen et al., 1999; Kunji et al., 1996; Pritchard and Coolbear, 1993).
The proteolytic systems of dairy LAB have received extensive research attention due to their importance in the physiology of these organisms and cheese flavor development. Because LAB are fastidious microorganisms with multiple amino acid auxotrophies (Kok and De Vos, 1994), during growth in milk, LAB rely on their proteolytic systems to obtain essential amino acids from caseins (CNs), the most abundant proteins in milk (Christensen et al., 1999; Kunji et al., 1996). In many cheese varieties, enzymatic conversion of large, casein (CN)-derived peptides into small peptides and free amino acids by LAB has pronounced effects on cheese flavor development as well as cheese functional properties. Additionally, proteolytic enzymes from LAB produce flavor compounds and precursors that are essential for cheese flavor development (Christensen et al, 1999; Mulholland, 1997).
Proteolytic systems of LAB can be functionally divided into three components: (i) cell envelope-associated proteinases which hydrolyze caseins to oligopeptides; (ii) peptide transport systems, of which the oligopeptide transport system is of greatest importance in milk and cheese; (iii) and numerous intracellular peptidases (Christensen et al., 1999; Kunji et al., 1996). The intracellular peptidases of LAB consist of both endopeptidases and aminopeptidases. Endopeptidases, due to their ability to hydrolyze peptide bonds within a peptide, are of particular interest in targeting peptides for rapid hydrolysis. Both the peptides αS1-CN(f1-9) and β-CN(f193-209), as well as other related hydrophobic peptide derivatives, are known to accumulate and have been associated with bitter defects in ripened cheeses (Broadbent et al., 2002; Broadbent et al., 1998; Exterkate and Alting, 1995; Kaminogawa et al., 1986; Lee et al., 1996; Lemieux and Simard, 1991). The peptide β-CN(f193-209) is produced by the activity of chymosin on β-CN.
Interest in the proteolytic system of L. helveticus CNRZ32 is related to the organism's ability to reduce bitterness and accelerate cheese flavor development when used as an adjunct culture in Gouda cheese production (Bartels et al., 1987a; Bartels et al., 1987b). The ability of Lb. helveticus CNRZ32 to accelerate cheese ripening and reduce bitterness when used as an adjunct culture is well documented (Bartels et al., 1987a; Bartels et al., 1987b; Madkor et al., 2000). Lb. helveticus CNRZ32 has been demonstrated to efficiently hydrolyze casein, and comparison with the peptidolytic activities of Lb. helveticus ATCC 10797 and Lactobacillus delbrueckii ssp. bulgaricus ATCC 12278 demonstrated that Lb. helveticus CNRZ32 had higher general aminopeptidase and dipeptidase activities (Khalid et al., 1991).
The reduction of bitterness in cheese is believed to be the result of preferential hydrolysis of low molecular weight hydrophobic peptides known to cause bitterness, rather than lack of formation of bitter peptides from high molecular weight non-bitter casein derived peptides (Broadbent et al., 1998; Gomez et al., 1996; Lee et al., 1996; Lemieux and Simard, 1991). These bitter peptides contain the amino acid proline, which forms an imino, not amino bond, making these peptides more difficult to cleave. While numerous enzymes of the proteolytic system of Lb. helveticus have been identified (Christensen et al., 1999), the understanding of the specific enzymes responsible for this strain's ability to reduce of bitterness in cheese is incomplete. Thus, identification and characterization of these enzymes are needed.
Moreover, endopeptidases in other contexts have also been explored. A prolyl endopeptidase was used to reduce the antigencity of a peptide involved in celiac sprue, an inflammation of the small intestine (Shan et al. 2002; Vader et al., 2002). Celiac sprue involves gluten peptides that survive the digestion process and reach the small intestine because they contain proline (see Schuppan et al., 2002). The use of other prolyl endopeptidases could provide therapeutic benefits for patients with celiac sprue.